Mitochondria are very metal rich. Precise levels of metals are required and typically bind to specific cognate proteins for optimal mitochondrial function. However during aging, genetic and epigentic changes occur that lead to decay in mitochondrial function, including altered metal homeostasis. Fluctuations in metal content and metalloprotein levels can cause subcellular and cellular dysfunction, and eventually disease. To clearly understand the relationship between metal homeostasis and mitochondrial function, a complete description and quantitation of all metals (the metallome) and all metalloproteins (the metalloproteome) in the mitochondria must be made available, yet technical obstacles have hindered progress towards this goal. Recently though, novel methods were developed that merge ultrasensitive elemental analysis with high- throughput proteomic tools resulting in the capacity to separate and identify hundreds of proteins and simultaneously reveal bound metals. This technology is optimally suited to quantify metals and identify metal-binding proteins within discrete proteomes, such as the mitochondria. By measuring the shifting patterns in metal content and metalloprotein expression patterns over time, a more comprehensive understanding of the effects of aging on mitochondrial function can be ascertained. In this preliminary study, (1) metal and metalloprotein content in mitochondria from the rat brain cortex will be quantitated, as cortical regions are a known target of aging and neurodegenerative disease. Then, (2) the hypothesis that mitochondrial metal homeostasis is altered during aging will be tested by mapping the major changes in metal content and metalloprotein patterns from young, middle-aged, and old adult rat cortex. Analysis of expression and elemental content will be performed on mitochondrial protein fractions separated by native liquid-phase isoelectric focusing followed by continuous elution electrophoresis. Protein mass and elemental content will be quantitated with scanning transmission ion microscopy and proton induced X-ray emission, respectively, and then combined with protein identification determined from MALDI-TOF and LC-TOF-MS analyses to generate the metallome and metalloproteome for rat cortical mitochondria. These results will be published as an Internet-based searchable database, highlighting age-related changes. Thus, this work has significant potential to integrate numerous aspects of mitochondrial metabolism and physiology and reveal novel opportunities for combating aging-related decline. [unreadable] [unreadable] [unreadable]